Project/Summary Vertebrate and invertebrate sleep are controlled by conserved signaling molecules, which suggests that sleep is evolutionarily ancient. Caenorhabditis elegans provides a powerful means for rapid identification and characterization of sleep-regulating pathways with single cell resolution because of their fast generation time and a simple and defined 302-celled nervous system. One pathway shown to regulate sleep in all animals involves cyclic adenosine monophosphate (cAMP), a ubiquitous second messenger that largely functions in neurons. C. elegans sleep in response to cellular stress caused by environmental insults (stress-induced sleep), an intriguing behavioral model for studying sleep during sickness. This project will define where cAMP is functioning in the C. elegans nervous system during stress-induced sleep. It will also determine where and how stress and sleep intersect in the nervous system. In Aim 1, we will identify the neurons in which cAMP is functioning and measure changes in cAMP levels in live animals while they sleep. To accomplish this, we will use a red light activated adenylyl cyclase that allows for cell-specific induction of cAMP in vivo and we will use a biosensor to measure cAMP changes in real-time during sleep. In Aim 2, we will test the hypothesis that the master stress sensor adenosine monophosphate-activated protein kinase (AMPK) is functioning in the same neurons as cAMP, downstream of protein kinase A, and is activated during sleep in response to cellular stress. In Aim 3 we will connect these newly identified sleep neurons to the known circuitry of stress induced sleep and test the hypothesis that AMPK is functioning downstream of PKA.